1. Technical Field of the Invention
The present invention relates generally to influenza vaccines, more specifically to avian influenza vaccines and formulations thereof useful for vaccinating susceptible avian species. The invention also relates to new methods for preventing or ameliorating avian influenza viral disease in poultry.
2. Description of the Related Art
Influenza viruses, most notably particular strains of A and B virus, are a serious cause of morbidity and mortality throughout the world, resulting in annual disease outbreaks. Periodically but at irregular intervals pandemics occur and result in particularly high levels of illness and death. Pandemics are historically the result of novel virus subtypes of influenza A, created by reassortment of the segmented genome (antigenic shift), whereas annual epidemics are generally the result of evolution of the surface antigens of influenza A and B virus (antigenic drift). Human influenza viruses often originate from avian strains of influenza virus so that influenza infection is at its basis a zoonosis. There is also evidence that swine can serve as an intermediate host (“mixing vessel”) for the generation of new avian-originated strains that are pathogenic in humans (Scholtissek et al., Virology 1985, 147:287). The H5N1 influenza A outbreak in Hong Kong in 1997 showed that highly pathogenic influenza A viruses can also be transmitted directly from avian species to humans (Claas et al., Lancet 1998, 351:472; Suarez et al., J. Virol. 1998, 72:6678; Subbarao et al., Science 1998, 279:393; Shortridge, Vaccine 1999, 17 (Suppl. 1): S26-S29). In 2003, the H5N1 viruses in Southeast Asia comprised different co-circulating geneotypes, but in 2004 a single genotype, known as the “Z-genotype”, became dominant (Li et al., Nature 2004, 430:209).
Current evidence indicates that fatal human cases resulted from the direct transmission of this genotype from birds to humans and that it also infected cats, with direct cat to cat transmission (Kuiken et al., Science 2004, 306:241). This and other evidence of the changing host range and widespread distribution of this virus raised concern that H5N1 viruses may acquire the characteristics that allow transmission from human to human. Humans would have no immunity to such new H5N1 viruses, which could cause catastrophic pandemic influenza (Fouchier et al., Nature 2005, 435:419). The potential of influenza A viruses to generate new pathogenic strains from a vast number of circulating strains in animal reservoirs indicates that disease control requires monitoring these viruses and developing improved antiviral therapies and vaccines. The speed with which new viral strains develop demands vigilance in this monitoring effort, including improved techniques for assessing the efficacy of vaccines to novel strains.
Avian Influenza, also called “AI,” is an acute and highly contagious viral infection of chickens and other fowl. As an influenza virus, it is classified in subtypes on the basis of antigen differences in the hemagglutinin (HA; also may be abbreviated as H) and neuraminidase (NA; also may be abbreviated as N) molecules, which “reassort” or “mutate” from season to season. Because the virus constantly mutates, it vaccine preparation is difficult due to the unpredictability as to which strain will reappear in subsequent seasons. The strains used for vaccine preparation often do not reproduce under manufacturing conditions at a very fast rate, so that waiting for an appearance of a particular strain, and then manufacturing the correct vaccine to protect against the strain does not provide a viable option. Typically, the epidemic of the particular strain will last for several months, and then perhaps disappear for several years.
Influenza viruses are classified into various A, B, and C topologies, according to the virus' group antigen. Influenza viruses of the A, B, and C types are distinguishable on the basis of antigenic differences in viral nucleocapsid (NP) and matrix (M) proteins. A-type influenza viruses are classified into subtypes on the basis of such differences in hemagglutinin (HA) and neuraminidase (NA). Nine subtypes of the neuraminidase NA proteins, designated NA 1 to NA 9, and fifteen different subtypes of the serum hemagglutinin HA proteins, designated HA 1 to HA 15, have been identified. In birds, viruses carrying each of the various HA (or H) and NA (or N) subtypes have been isolated.
Influenza A, B and C, of the family Orthomyxoviridae, all have a segmented negative strand RNA genome that is replicated in the nucleus of the infected cell, has a combined coding capacity of about 13 kb, and contains the genetic information for ten viral proteins. Specifically, influenza viruses have eight negative-sense RNA (nsRNA) gene segments that encode at least 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (HA, which after enzymatic cleavage is made up of the association of subunits HA1 and HA2), the matrix proteins (M1 and M2) and the non-structural proteins (NS1 and NS2) (Krug et al., In The Influenza Viruses, R. M. Krug, ed., Plenum Press, New York, 1989, pp. 89-152).
Recently developed reverse-genetics systems have allowed the manipulation of the influenza viral genome (Palese et al., Proc. Natl. Acad. Sci. USA 1996, 93:11354; Neumann and Kawaoka, Adv. Virus Res. 1999, 53:265; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al., J. Virol. 1999, 73:9679). For example, it has been demonstrated that the plasmid-driven expression of eight influenza nsRNAs from a pol I promoter and the coexpression of the polymerase complex proteins result in the formation of infectious influenza A virus (Hoffmann et al., Proc. Natl. Acad. Sci. USA 2000, 97:6108).
The virus particle of the influenza virus has a size of about 125 nm and consists of a core of negative sense viral RNA associated with the nucleoprotein, surrounded by a viral envelope with a lipid bilayer structure. The inner layer of the viral envelope is composed predominantly of matrix proteins and the outer layer contains most of the host-derived lipid material. The so-called “surface proteins”, neuraminidase (NA) and hemagglutinin (HA), appear as spikes on the surface of the viral body. Infectivity of novel influenza viruses depends on the cleavage of HA by specific host proteases, whereas NA is involved in the release of progeny virions from the cell surface and prevents clumping of newly formed virus.
The HA and NA proteins embedded in the viral envelope are the primary antigenic determinants of the influenza virus (Air et al., Structure, Function, and Genetics, 1989, 6:341-356; Wharton et al., In The Influenza Viruses, R. M. Krug, ed., Plenum Press, New York, 1989, pp. 153-174). Due to reassortment of influenza segmented genome, new HA and NA variants are constantly created for which a newly infected organism has no anamnestic immune response. HA glycoprotein is the major antigen for neutralizing antibodies and is involved in the binding of virus particles to receptors on host cells.
HA molecules from different virus strains show significant sequence similarity at both the nucleic acid and amino acid levels. This level of similarity varies when strains of different subtypes are compared, with some strains clearly displaying higher levels of similarity than others (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643). The levels of amino acid similarity vary between virus strains of one subtype and virus strains of other subtypes (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643). This variation is sufficient to establish discrete subtypes and the evolutionary lineage of the different strains, but the DNA and amino acid sequences of different strains are still readily aligned using conventional bioinformatics techniques (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643; Suzuki and Nei, Mol. Biol. Evol. 2002, 19:501).
HA is a viral surface glycoprotein comprising approximately 560 amino acids and representing 25% of the total virus protein. It is chiefly responsible of adhesion of the viral particle to and its penetration into a host cell in the early stages of infection. Among the viral proteins, hemagglutinin is most subject to post-translational rearrangement. After synthesis of hemagglutinin has been completed, the molecule follows the exocytotic pathway of the host cell, in the course of which HA is folded, assembled in trimers and glycosylated. Finally, HA is cleaved into two subunits Hi and H2; which activates the molecule and promotes the virion's infective capacity.
Differences in the sequence of basic amino acids within the cleavage site correlates with the capacity of the avian influenza virus to produce localized, symptomatic infections or generalized infections having a lethal outcome for many avian species. It has therefore been suggested that this feature might be important in influencing the virus' organ-tropism, host specificity, as well as its pathogenicity. With respect to the pathogenicity of the virus, strains with multibase-site HA find proteases that cleave the HO molecule, in the active form H1 and H2 in several cellular types thus giving rise to multiple infection cycles with a massive production of infectious viral particles and causing a generalization of the infections in all of the districts within a short time period (HPAI strains). The infection will consequently turn out to have an acute-hyperacute course, with very high mortality.
Neuraminidase (NA) is a second membrane glycoprotein of the influenza A viruses. NA is a 413 amino acid protein encoded by a gene of 1413 nucleotides. NA participates in the destruction of the cellular receptor for the viral hemagglutinin by cleaving between the sialic acid molecule and the hemagglutinin itself. In this way it believed to be possible to ease liberation of viral progeny by preventing newly formed viral particles from accumulating along the cell membrane as well as by promoting transportation of the virus through the mucus present on the mucosal surface. NA is an important antigenic determinant that is subject to antigenic variations.
The influenza vaccines currently licensed by public health authorities for use in the United States and Europe are inactivated influenza vaccines as well as the live attenuated FLUMIST vaccine in the United States. Viruses presenting epidemiologically important influenza A and influenza B strains are grown in embryonated chicken eggs and the virus particles are subsequently purified and inactivated by chemical means to form vaccine stocks. Each year the WHO selects subtypes which most likely will circulate for that year for vaccine development.
Although influenza vaccines have been in use since the early 1940's for human vaccination and since the late 1960's for equine vaccination, the existence of extensive animal reservoirs, combined with the threat of emergence of a novel influenza virus capable of causing a pandemic, has spurred research into novel therapies with which to fight the virus. Several important advances in the field of influenza have occurred in the last few years (reviewed in Cox and Subbarao, Lancet 1999, 354:1277-82). For example, an experimental live, attenuated, intranasally administered trivalent influenza vaccine was shown to be highly effective in protecting young children against influenza A H3N2 and influenza B. Other approaches to improve the efficacy of the current (killed) influenza virus vaccines include the generation of cold-adapted and genetically engineered influenza viruses containing specific attenuating mutations (reviewed in Palese et al., J. Infect. Dis., 1997, 176 Suppl 1:S45-9). It is hoped that these genetically altered viruses, in which the HA and NA genes from circulating strains have been incorporated by reassortment, can be used as safe live influenza virus vaccines to induce a long-lasting protective immune response in humans. Although cold-adapted vaccines appear to be efficacious in children and young adults, they may be too attenuated to stimulate an ideal immune response in elderly people, the major group of the 20000-40000 individuals in the USA dying each year as a result of influenza infection.
Readily available vaccines would provide the most effective tool against emergent pandemic influenza. After the 1997H5N1 outbreak in Hong Kong, vaccines produced by two different approaches were tested in humans. Conventional subunit H5 vaccine produced from A/duck/Singapore/3/97 was poorly immunogenic in humans, even against antigenically closely related strains and after multiple vaccination (Nicholson et al., Lancet 2001, 357:1937; Stephenson et al., Journal of Infectious Disease 2005, 191:1210). The use of the adjuvant MF59 increased the antibody titer of this H5 vaccine (Stephenson et al., Vaccine 2003, 21:1687). Vaccination with inactivated “split” vaccine derived from nonpathogenic A/duck/HK/836/80 (H3N1) virus and the modified H5 hemagglutinin from A/HK/156/97 (H5N1) virus induced barely detectable titers of neutralizing antibodies (Takada et al., Journal of Virology 1999, 73:8303). Thus, although these H5N1 vaccines were well tolerated, they appeared to be poorly immunogenic. The current lack of effective vaccines against H5N1 virus strains increases the threat of these viruses to cause pandemic disease.
Serum antibody titer methods are the accepted surrogate measures of immune protection after vaccination or viral infection. The predominantly used serum antibody titer methods are virus neutralization titer assays and hemagglutinin inhibition (HI) titer assays. These assays are based on the ability of influenza antibodies from human serum to cross react with antigens under in vitro conditions. Assays are selected for a given situation based not only on their ability to provide consistent and applicable results but also based on their ease of use and the facility requirements for each type of assay.
Briefly stated, the virus neutralization assay examines the ability of antibodies from a serum sample to block the infection of cultured cells by influenza virus. The assay is carried out by creating serial dilutions (titers) of a serum sample and combining each of these dilutions with a standard amount of infectious virus. Each dilution mixture is then presented to a defined cell culture and the resulting infection rates assayed. The virus neutralization titer assay is considered to be an extremely useful and reliable test to examine the level of immunoprotective antibodies present in a given individual. It is, however, dependent on specialized cell culture facilities and therefore is not universally available. The methodology is also laborious and time consuming hence poorly suited to screening large numbers of samples.
The hemagglutinin inhibition (HI) assay similarly examines the ability of antibodies from a serum sample to bind with a standardized reference virus. The basis for this assay is the fact that influenza viruses will bind to and agglutinate erythrocytes. In the HI assay, serial dilutions of serum sample are mixed with standard amounts of reference virus and after a set incubation period added to erythrocytes. The association between reference viruses and erythrocytes into complexes is then detected visually. The highest dilution of serum that inhibits hemagglutinin is read as the hemagglutinin inhibition titer. Although not as sensitive of vaccine immunogenicity as other assays, the HI assay is widely employed due to its relatively simple technology and laboratory requirements.
The current Asian H5N1 highly pathogenic avian influenza has spread over much of Asia and into Europe and Africa. As well as affecting village and commercial chicken operations in many South East Asian countries, it differs from past H5 avian influenzas in that it causes morbidity and mortalities in other domesticated birds, such as ducks and turkeys and in wild waterbirds. Effective vaccines that can prevent infection, as well as disease, and be used in a variety of avian species are needed for field use.
The major control strategy for highly pathogenic avian influenza (HPAI) outbreaks in poultry has traditionally been one of eradication via movement restrictions and slaughter of affected and at-risk birds. With the widespread presence, however, of the current Asian H5N1 virus in village poultry, including ducks and turkeys, and in wildlife species, particularly migrating birds, alternate control strategies must be considered, with vaccines likely to be a key component.
Currently available commercial vaccines for avian influenza are oil emulsion killed virus vaccines that have mostly been used to control endemic low pathogenic avian influenza (LPAI) in chickens and turkeys, or HPAI outbreaks in Pakistan and Mexico. Halvorson, Avian Path. 31:5-12 (2002); Naeem, Proceedings of the 4th International Symposium on Avian Influenza 31-35. (Athens, Ga., USA, 1998); Swayne and Suarez, Rev. Sci. Tech. Off. Int. Epiz. 19:463-482 (2000). Both killed vaccines and recombinant fowlpox vaccines are currently used to control mildly pathogenic avian influenza in Mexico. Id. The European Union has approved use of inactivated oil emulsion vaccines for use in Italy, provided they allow for differentiation of vaccinated versus infected birds. Capua et al., Avian Path. 32:47-55 (2002).
While it has been demonstrated that an inactivated oil emulsion vaccine could interrupt transmission of H7N7 (Van der Goot et al., Proc. Natl. Acad. Sci. U.S.A. 102:18141-18146 (2005)) or the current H5N1 HPAI (Ellis et al., Avian Path. 33:405-412 (2004)), concerns still exist that these vaccines may not have 100% efficacy in the field and will not totally prevent shedding of virus. Additionally, their use does not allow the differentiation between vaccinated and infected birds, which interferes with monitoring the disease status within flocks and regions; nor have they been formulated or tested for vaccination efficacy in ducks.
The Asian H5N1 virus cannot be grown to high titer in eggs, which is the traditional method of virus production for human and avian influenza vaccines. Thus, alternatives to homologous virus vaccines are being developed. Live vectored vaccines can provide additional safety and the ability to differentiate between infected and vaccinated birds.
Fowlpox virus (Qiao et al., Avian Path. 32:25-31 (2003), infectious laryngotracheitis virus (Luschow et al., Vaccine 19:4249-4259 (2001) and adenovirus (Gao et al., J. Virol. 80:1959-1964 (2006)) vectors expressing H5 have all been assessed and shown to have protective efficacy, with reduction, but not complete elimination, of virus shedding. Other influenza strains, particularly those that share the H5 hemagglutinin type, but with a different neuraminidase, have been shown to have some efficacy in the field (Ellis et al., Avian Path. 33:405-412 (2004)), but the most promising approach is the use of reverse genetics to create an influenza virus that has the current H5 in a genetic background that allows growth to high titer in eggs, but has low pathogenicity in either avian or mammalian species.
Reverse genetics has been used to create influenza virus reassortants with the hemagglutinin and neuraminidase genes from either the human H5N1 isolate, A/HK/491/97 (Subbarao et al., Virol. 305:192-200 (2003)), or the avian H5N1 isolate, A/Goose/Guangdong/96 (Tian et al., Virol. 341:153-162 (2005). Both of these reassortants are apathogenic in chickens and the reassortant virus with H5 and N1 from A/Goose/Guangdong/96 has been tested as a formalin inactivated preparation for protective efficacy against the parent HPAI H5N1 in specific pathogen free (SPF) chickens, and in non-SPF geese and ducks. Those studies demonstrated that the reassortant vaccine could prevent mortality and reduce shedding of the challenge virus.
Inclusion of a different neuraminidase subtype in the reassortant vaccine allows differentiation of infected from vaccinated birds. This principle has been demonstrated with reassortants using hemagglutinin genes from H5 and H7 LPAI viruses and the remaining genes, including the N1 from A/WSN/33 (Lee et al., Vaccine 22:3175-3181 (2004)). Oil emulsion vaccines with these reassortants reduced replication of the parental H5 and H7 LPAI strains in SPF chickens. A reassortant virus with H5 from A/Goose/Hong Kong/437-4/99 and N3 from A/Duck/Germany/1215/73 has been constructed (Liu et al., Virol. 314:580-590 (2003)). When formulated as an oil emulsion, the vaccine was able to protect SPF chickens against mortality following challenge with HPAI H5N1 virus. At appropriate doses of the vaccine, there was no challenge viruses detected in the birds.
Reverse genetics influenza vaccines have been postulated to be utilizable in these “DIVA” methods, where a vaccine is administered having an N different from the viral strain against which the bird is being vaccinated thereby facilitating the differentiation of vaccinated animals from infected birds. Published PCT WO 03/086453, which is incorporated by reference in its entirety herein, describes the DIVA technology, and some representative vaccines utilizable in the methods thereof.
Reverse genetics vaccines offer a number of obvious advantages over conventional vaccines prepared from naturally occurring virus strains. Through reverse genetics technology, specific genes from virus A may be replaced with the corresponding gene from virus B. Additionally, these genes may be modified to reduce viral pathogenicity while retaining the resulting vaccine's protective properties.
Vaccines based on the current Asian H5N1 strain and which overcome these problems and for use in a variety of poultry species are urgently needed that provide an alternative to eradication of infected flocks. A requirement of such avian influenza virus vaccines is that they (a) elicit a rapid immune response in the vaccinated avian and (b) enable differentiation of vaccinated birds from infected birds. Thus, there remains a need for improved avian influenza vaccines which not only invoke a rapid immune response, and a higher titer response, but which also produce a sterilizing effect, preventing the growth, shedding and transmission of a challenge virus to other susceptible species.